PROJECT SUMMARY/ABSTRACT Colorectal cancer (CRC) is currently the second leading cause of cancer deaths in the United States and, as of 2018, shows an increasing mortality rate in younger age groups. The reason for this rise in mortality has not been fully explained and highlights the urgent need to better understand the causes and risk factors for CRC and develop novel strategies for its prevention and treatment. A growing body of evidence has implicated members of the human gut microbiome as potential drivers of CRC development. In particular, bacteria that produce a small molecule genotoxin known as colibactin may be key players in this process. Colibactin is produced by both commensal and pathogenic organisms which harbor the pks genomic island. Numerous studies have shown that transient infection of mammalian cells with pks+ E. coli leads to DNA crosslinking, DNA double-strand breaks, chromosomal instability, and senescence. Clinical studies have shown that pks+ bacteria are more prevalent in patients with CRC (~68%) and IBD (~40%) in comparison to healthy controls (21%) and are more abundant in tumor tissue biopsies than those from adjacent healthy tissue. In animal models, colonization with pks+ E. coli in a mouse model of CRC leads to increased tumor load relative to mice colonized with non-colibactin-producers. In perhaps the most direct evidence of colibactin?s carcinogenic potential, recent work has also shown that when mice are colonized with pks+ E. coli, colibactin directly alkylates DNA in gut epithelial cells, resulting in the formation of DNA adducts. Together, this evidence suggests that exposure to colibactin may increase risk for, or accelerate the development of, CRC via the mutagenic effects of DNA alkylation and crosslinking by colibactin. Despite this, no viable therapeutic strategy has emerged to prevent colibactin exposure, and no tools exist to study the effects of this pathway in the context of a complex, healthy gut-microbial community. This proposal aims to address these knowledge gaps by developing potent and specific small molecule inhibitors of colibactin biosynthesis. Such tool compounds will enable a more detailed study of how colibactin contributes to CRC progression and allow us to test the hypothesis that blocking colibactin production by pks+ bacteria using small molecules can lower the risk of developing CRC for the host. This research will both to explore a novel molecular target for therapeutic intervention in CRC development, as well as provide the wider scientific community with tools that will enable a more precise study of the impacts of small molecule toxins from commensal microbes on human health.